7. PROJECT SUMMARY / ABSTRACT Chemical modification of mRNA provides a powerful means to dynamically alter gene expression in eukaryotes via epitranscriptomic changes. In particular, methylation of adenosine at the N6 position (m6A) constitutes the most widespread internal base modification to mRNA. Modification of mRNA by m6A influences numerous biological processes including development, differentiation, reprogramming, circadian rhythm, cell cycle, disease pathogenesis, and stress responses including virus infection. Significantly, virus-encoded mRNAs are also chemically modified by m6A, and a role for m6A in Human Cytomegalovirus (HCMV) infection biology is emerging. As a canonical TORCH (T. gondii, other, rubella virus, HCMV, HSV) pathogen, primary HCMV infection during pregnancy remains the leading viral cause of birth defects. While HCMV infection causes mild if any maternal morbidity and is predominately asymptomatic in healthy individuals, it results in life-threatening disease among the immunocompromised, including solid-organ or stem cell transplant recipients, and is a significant source of congenital morbidity and mortality among newborn infants in the developed world. Addressing HCMV congenital infection remains a serious unmet medical need as there is no HCMV vaccine to prevent primary infection during pregnancy and no current treatment to prevent transmission from mother to fetus. Our long-term objective is to understand how the chemical modification of host and/or viral RNA by m6A impacts reproduction of HCMV, a common infection that remains the leading viral cause of congenital abnormalities. Preliminary results demonstrate that cellular m6A methyltransferase subunits METTL3/14, the m6A demethylase ALKBH5, and m6A recognition proteins regulate HCMV reproduction and responses to double strand DNA (dsDNA) in uninfected cells. This is achieved in part through changes in interferon b gene (IFNB1) expression. These findings establish that m6A RNA modification enzymes regulate cellular responses to HCMV and dsDNA sensing, which shapes host immunity and contributes to autoimmune disease. It further suggests that m6A epitranscriptomic changes play a fundamental role in cell-intrinsic innate immune responses to the TORCH pathogen HCMV. Based upon our preliminary results, we hypothesize that HCMV reproduction is differentially controlled by the host m6A modification machinery. Here, this hypothesis will be tested in three specific aims designed to: (i) identify how the host m6A modification machinery is regulated in response to HCMV infection; (ii) determine how cellular m6A modification enzymes regulate IFNB1 mRNA accumulation in HCMV-infected cells; and (iii) identify how HCMV gene expression is impacted by differential m6A modification. The project is significant because it investigates how epitranscriptomic changes impact HCMV reproduction and innate immunity. Understanding how HCMV infection is regulated by epitranscriptomic RNA modification could lead to new opportunities for therapeutic intervention and possibly new strategies for vaccine development.